Antibodies are essential tools for experimental research, diagnostics, and therapeutic applications. Monoclonal antibodies have revolutionized biotechnology and are now key therapeutic drugs in the treatment of human disease. Despite their successes, therapeutic monoclonal antibodies have certain limitations, such as restricted activity against certain types of antigen, poor tissue penetration, unwanted effector function in many situations, the cost of manufacturing, product instability and aggregation.
Conventional antibody molecules are composed of two heavy chain polypeptides linked to two light chain polypeptides by disulphide bridges. The combined variable regions of the heavy and light chains define the binding site by which the antibody interacts with its cognate antigen. In addition to these conventional antibodies, camelids and sharks produce another class of functional immunoglobulins, which are composed of heavy chains only. These heavy-chain only antibodies are naturally devoid of light chains, and can bind their cognate antigen using a single domain. The antigen binding surface of these single-chain antibodies is usually more convex (or protruding) than the one of conventional antibodies, which is usually flat or concave.
The identification of smaller binding proteins that retain high specificity and affinity for the target protein would be beneficial for access to hard-to-reach antigens. Single domain antibodies that occur naturally in the shark are particularly attractive for the development of next generation biotherapeutics. IgNARs (Immunoglobulin New Antigen Receptors) heavy chain-only Ig-like molecules have been identified in all species of sharks studied so far. They are disulphide-bound homodimeric molecules composed of two polypeptide chains containing five constant domains and one variable region (VNAR) by which they bind antigens (Greenberg et al., Nature 1995 Mar. 9; 374(6518):168-73).
VNARs are small (12 kDa), stable, soluble, monomeric antigen-binding domains that can be configured into many different therapeutic modalities. The isolation of various VNAR based binding moieties has been described (see, e.g., WO2003/014161 and WO2005/118629). Owing to their elongated CDR3 structures that potentially extend into antigen clefts and cavities, VNARs are well suited to the purpose.
The VNAR protein scaffold consists of amino acid residues (aa) 1-25 of the framework 1 (FW1) region; aa 26-32 of the complimentary determining region 1 (CDR1); aa 33-43 of FW2; aa 44-52 of the hypervariable 2 region (HV2); aa 53-85 of FW3; aa 61-65 of HV4; the CDR3 region (of variable length) and FW4 (11 residues starting at XGXG); see FIGS. 1A and B. Like all immunoglobulin family variable (V) domains, VNARs contain the two canonical cysteine residues that link FW1 and FW3 via a disulfide bond. Additionally, VNARs contain non-canonical cysteines which define two different structural isotypes. Type 1 VNARs contain two cysteine residues in CDR3, which form disulphide bridges to non-canonical cysteine residues in FW2 and FW4. In addition, Type 1 VNARs may also contain an even number of extra cysteines in CDR3, which form intraloop cysteine bridges. Type 2 VNARs contain only a single extra disulphide bond, which links CDR1 and CDR3. (FIG. 1A)
Regardless of the VNAR isotype, CDR1 and CDR3, and to a lesser extent HV2 and HV4, show a high level of sequence variability and are considered the major determinants for antigen binding. Some clones however, were shown to recognize their cognate antigen by also making a number of contacts outside of the CDRs. A high-affinity human serum albumin (HSA)-binding VNAR isolated from spiny dogfish, for example, was shown to interact with HSA in an atypical manner by making several framework contacts in addition to contacts to CDRs (Kovalenko et al. J Biol Chem. 2013 Jun. 14; 288(24):17408-19).
In order to circumvent the limitations of developing VNARs in living animals, several synthetic phage display libraries have been generated based on VNAR backbones from different shark species (see e.g., Nuttall et al., Eur J Biochem. 2003 September; 270(17):3543-54; Shao et al., Mol Immunol. 2007 January; 44(4):656-65. Epub 2006 Feb. 24). All these libraries, however, are based on CDR3 randomization of a single VNAR clone. It would thus be beneficial to have new VNAR libraries characterized by higher overall sequence diversity through CDR randomization in the context of a plurality of VNAR framework sequences from which high affinity binding proteins to molecular targets may be selected.